1. Field of the Invention
The present invention relates to an apparatus for performing biological reactions. Specifically, the invention relates to an apparatus for performing nucleic acid hybridization reactions on a substrate layer having a multiplicity of oligonucleotide binding sites disposed thereon.
2. Description of the Prior Art
Recent advances in molecular biology have provided the opportunity to identify pathogens, diagnose disease states, and perform forensic determinations using gene sequences specific for the desired purpose. This explosion of genetic information has created a need for high-capacity assays and equipment for performing molecular biological assays, particularly nucleic acid hybridization assays. Most urgently, there is a need to miniaturize, automate, standardize and simplify such assays. This need stems from the fact that while these hybridization assays were originally developed in research laboratories working with purified products and performed by highly skilled individuals, adapting these procedures to clinical uses, such as diagnostics, forensics and other applications, has produced the need for equipment and methods that allow less-skilled operators to effectively perform the assays under higher capacity, less stringent assay conditions.
Nucleic acid hybridization assays are advantageously performed using probe array technology, which utilizes binding of target single-stranded DNA onto immobilized DNA (usually, oligonucleotide) probes. The detection limit of a nucleic acid hybridization assay is determined by the sensitivity of the detection device, and also by the amount of target nucleic acid available to be bound to probes, typically oligonucleotide probes, during hybridization.
Nucleic acid hybridization chambers are known in the prior art.
U.S. Pat. No. 5,100,755 to Smyczek et al. discloses a hybridization chamber.
U.S. Pat. No. 5,545,531 to Rava et al. discloses a hybridization plate comprising a multiplicity of oligonucleotide arrays.
U.S. Pat. No. 5,360,741 to Hunnell discloses a gas heated hybridization chamber.
U.S. Pat. No. 5,922,591 to Anderson et al. discloses a miniaturized hybridization chamber for use with oligonucleotide arrays.
U.S. Pat. No. 5,945,334 to Besemer discloses oligonucleotide array packaging.
As currently employed, oligonucleotide array technology does not provide maximum hybridization efficiency. Existing nucleic acid hybridization assay equipment includes numerous components, each of which is a source of inefficiency and inaccuracy.
Hybridization using oligonucleotide arrays must be performed in a volume in which a small amount of target DNA or other nucleic acid can be efficiently annealed to the immobilized probes. For diagnostic assays, target DNA molecules are often obtained in minute ( less than picomol) quantities. In practice, it can take several (tens of) hours for hybridization to be substantially complete at the low target nucleic acid levels available for biological samples.
In addition, array hybridization is conventionally performed in a stationary hybridization chamber where active mixing is absent. Under these conditions, the probability that a particular target molecule will hybridize to a complementary oligonucleotide probe immobilized on a surface is determined by the concentration of the target, the diffusion rate of the target molecule and the statistics of interaction between the target and the complementary oligonucleotide.
Consequently, a larger number of samples must be tested to obtain useful information, and this in turn leads to increased hybridization times and inefficiencies.
In addition, efficiency is increased when the amount of user manipulation is kept to a minimum. As currently performed, oligonucleotide array hybridization requires a great deal of operator attentiveness and manipulation, and the degree of skill required to perform the analysis is high. For example, hybridization is typically performed in an assay chamber, and then data collection and analysis are performed in a separate apparatus (such as a laser scanner or fluorescence microscope). This arrangement requires a substantial amount of handling by the user, and makes the assays both time-consuming and subject to user error.
It is also a limitation of current practice that array hybridizations are performed one array at a time, thereby forgoing the economies of parallel processing and data analysis.
Additional limitations, inefficiencies, and expenses arise from the structural characteristics of existing apparatus. Many existing apparatus are limited in the size of the substrate they can accommodate. Other apparatus are not disposable and therefore require extensive cleaning between runs in order to prevent sample contamination. Yet other apparatus are high mass and therefore not susceptible of the rapid heating and cooling necessary for efficient hybridization. Other apparatus require the use of expensive optics for analysis of the reaction products.
There remains a need in this art for an easy-to-use apparatus for performing biological reactions, particularly nucleic acid hybridization, that comprises a small reaction volume, where the fluid components can be actively mixed, that can be performed in parallel and that minimizes user intervention. There also remains a need for such an apparatus that is easy to manufacture in various sizes, that is disposable to minimize sample contamination, that allows for the use of low cost optical analytical equipment, and that is low mass to allow for rapid heating and cooling of the sample fluid. There also remains a need for methods for using such apparatus to increase hybridization efficiency, particularly relating to biochip arrays as understood in the art. This need is particularly striking, in view of the tremendous interest in biochip technology, the investment and substantial financial rewards generated by research into biochip technology, and the variety of products generated by such research.
The invention provides an apparatus in which biological reactions such as nucleic acid hybridization can be performed. The apparatus of the invention is a hybridization chamber comprising a flexible layer attached to a biochip by an adhesive layer. The biochip comprises a substrate having a first surface and a second surface, wherein the first surface contains an array of biologically reactive sites, preferably an oligonucleotide array. The array is provided in an area bounded by the adhesive layer set down on the first substrate surface. The flexible layer most preferably is both deformable and translucent. The chamber also includes a first port, and certain embodiments further include a second port, that transverses the substrate and comprises a first opening on the first substrate surface and a second opening on the second substrate surface. The openings of these ports on the second substrate surface are covered by a removable cover, most preferably a foil tape. The openings of these ports on the first substrate surface are provided within the area bounded by the adhesive layer. The adhesive layer, the flexible layer and the substrate also define a volume that is filled with a water-soluble compound. The water-soluble compound is preferably a solid at certain temperatures, most preferably at or below room temperature, and a liquid at elevated temperatures, most preferably below about 100xc2x0 C.
In certain embodiments of the invention, the substrate comprises a multiplicity of oligonucleotide arrays, which are contained in one or a plurality of areas bounded by the adhesive layer and covered by the flexible layer. In these embodiments, each area bounded by the adhesive layer also comprises a first port and can also comprise a second port.
The hybridization chamber is optionally supplied with a heater, most preferably a resistive heater, in thermal contact with the flexible layer. The chamber is also optionally supplied with a roller, most preferably a patterned roller, positioned in contact with the flexible layer and movable longitudinally across the surface of the chamber for mixing hybridization fluid and wash solutions as required. The chamber is further optionally supplied with a scanner positioned in contact with the flexible layer and movable longitudinally across the surface of the chamber for detecting hybridization signals, most preferably produced by fluorescently-labeled target nucleic acid.
In a further preferred embodiment, the invention provides a handheld device comprising a lid, a base, a carriage embodying a roller, a scanner, a heating element and a thermocouple, wherein the hybridization chamber of the invention is positioned above and in removable contact with the carriage.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.